The present invention is related to polynucleotide molecules and to their use for production of desired products after introduction into human or animal cells. In addition, the present invention is concerned with pharmaceutical compositions comprising said polynucleotide molecules and their use in prophylactic or therapeutic treatment methods. The present invention is also related to use of such polynucleotide molecules in animals to achieve expression of desired products, which can be recovered from the animal but do not give rise to any beneficial, e.g. therapeutical, activity in the said animal.
1. Field of the Invention
More specifically, the present invention is directed to alphavirus cDNA vectors comprised of recombinant cDNA consisting of cDNA derived from an alphavirus and heterologous, i.e. foreign, cDNA coding for a desired substance.
2. Description of Related Art
Alphavirus is a genus belonging to the family Togaviridae having single stranded RNA genomes of positive polarity enclosed in a nucleocapsid surrounded by an envelope containing viral spike proteins.
The Alphavirus genus comprises among others the Sindbis virus, the Semliki Forest virus (SFV), the Ross River virus and Venezuelan, Western and Eastern equine encephalitis viruses, which are all closely related. In particular, the Sindbis and the Semliki Forest viruses have been widely studied and the life cycle, mode of replication, etc, of these viruses are well known and thus, need not to be specifically discussed herein.
Alphaviruses replicate very efficiently in animal cells which makes them valuable as vectors for production of protein and nucleic acids in such cells.
Expression systems based on the Sindbis virus are disclosed in U.S. Pat. Nos. 5,091,309 an 5,217,879. The Sindbis virus vectors of U.S. Pat. No. 5,091,309 comprise RNA derived from Sindbis defective interfering (DI) RNA having heterologous RNA inserted therein.
In U.S. Pat. No. 5,217,879 self-replicating and self-packaging recombinant Sindbis virus RNA molecules are disclosed comprising a heterologous coding sequence and at least one Sindbis virus junction region able to direct Sindbis virus subgenomic messenger RNA synthesis in a host cell. RNA transcripts are synthesized in vitro by transcription of Sindbis virus cDNA which has been inserted in a plasmid under control of a promoter, such as SP6. The SP6 promoter and other promoters disclosed in connection with cDNA transcription are not functional in animal or human cells.
In WO 92/10578 (Garoff and Liljestrxc3x6m) an expression system based on alphaviruses is disclosed. An illustrative example of such viruses is the Semliki Forest virus (SFV). Earlier it was reported that a full-sized cDNA copy of the SFV RNA genome was contructed (Journal of Virology, Volume 65, pages 4107-4113, 1991). This was engineered into an SP6 transcription vector from which full-sized SFV genomic RNA molecules can be transcribed in vitro. The RNA can be transfected into animal cells, in which cells the RNA molecules will support normal wild-type virus infection, since the RNA molecules are of positive polarity and can function as messenger RNA molecules in the cells. Upon transfection, the first portion of the genome is translated into a polyprotein which self-cleaves into four non-structural proteins (nsP1-nsP4). These proteins constitute the alphavirus replicase and are responsible for the production of new full-length genomic RNA molecules as well as of a subgenomic RNA species starting from an internal promoter (26S promoter). They are also responsible for the capping of the 5xe2x80x2 end of the new RNA molecules. The pSFV4 cDNA plasmid was further engineered into a general DNA expression plasmid by deleting portions of the coding region for the structural proteins and replacing such deleted portions with a linker region for insertion of foreign coding sequences (Bio/Technology, Volume 9, pages 1356-1361, 1991; Bio/Technology, Volume 11, pages 916-920, 1993). When foreign DNA coding sequences are inserted into these vectors, high amounts of foreign protein are obtained when virus structural proteins are translated from the RNA subgenome made by the alphavirus replicase.
According to WO 92/10578, an RNA molecule is provided, which is derived from an alphavirus RNA genome and is capable of efficient infection of animal cells, which RNA molecule comprises the complete alphavirus genome regions, which are essential for replication of the said alpha-virus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof. According to WO 92/10578 such RNA molecules can be transferred into animal cells by any means of transfection or by packaging of said RNA molecules into infectious alphavirus particles for later infection of animal cells. In both cases the transfected or infected RNA molecule will be able to replicate within the target animal cell and to express the exogenus RNA sequences inserted into said RNA molecule. Such molecules and strategies for their expression within the cell can be used as vaccines or strategies to vaccinate in order to prevent or treat infection or cancer.
Since it is difficult to engineer RNA molecules by current genetic engineering technology, manipulations of the Alphavirus genome, such as insertion of heterologous coding sequences, have been conducted on the corresponding cDNA molecule. Subsequently, the engineered cDNA molecule has been transcribed in vitro and the RNA transcripts obtained have been used to transform cells. These constructs comprising the engineered cDNA molecule cannot be transcribed in animal or human cells since the promoters used for transcriptional control is not functional in such cells.
Obviously, it would be to advantage if the cDNA molecule could be used per se to transform cells and achieve expression of a desired substance in these cells.
WO 90/11092 describes the use of naked polynucleotides as a pharmaceutical which operatively codes for a biologically active peptide. Such molecules are proposed to be injected into tissue for the in vivo expression of said peptide. Specifically, it is claimed that the polynucleotide is DNA and that the peptide may function as an antigen, and may thus be used as a vaccine (see also Science, Volume 259, pages 1745-1749; DNA and Cell Biology, Volume 12, number 9, entire volume, 1993). However, recombinant viral cDNA constructs comprising heterologous coding sequences which can be expressed in animal and human cells are not disclosed, therein, nor is a cDNA construct disclosed, which is transcribed into self-replicating RNA encoding the replicase necessary for its replication. Even though, use of DNA coding for a polypeptide and for a polymerase for transcribing the DNA is disclosed in WO 90/11092, the initial quantity of polymerase is provided by including mRNA coding therefore in the preparation, which mRNA is translated by the cell.
Thus, it is an object of the present invention to provide a recombinant cDNA molecule complementary to an alphavirus RNA and comprising an exogenous cDNA sequence, which molecule can be introduced into animal or human cells to achieve transcription or expression of said cDNA, desired products such as polynucleotides or proteins being produced in cells harbouring the cDNA-molecule.
In accordance with the present invention, this object is achieved by placing the complete cDNA molecule under transcriptional control of a promoter sequence functional in an animal or human cell. Said promoter sequence will initiate transcription by the DNA-dependent RNA polymerase encoded by the host cell, i.e. the animal or human cell harbouring the said cDNA molecule.
Accordingly, the present invention is concerned with a cDNA molecule complementary to at least part of an alphavirus RNA genome, which CDNA molecule comprises the complement of the complete alphavirus RNA genome regions, which are essential for replication of the said alphavirus RNA, and further comprises an exogenous cDNA sequence capable of expressing its function in an animal or human host cell, said exogenous cDNA sequence being inserted into a region of the cDNA molecule, which is non-essential to replication thereof, and said cDNA molecule being placed under transcriptional control of a promoter sequence functional in said animal or human cell.
The promoter sequence of the present invention may comprise a promoter of eukaryotic or prokaryotic origin. The promoter region may also include control elements for repression or enhancement of transcription. Suitable promoters are the cytomegalovirus immediate early promoter (pCMV) and the Rous sarcoma virus long-terminal repeat promoter (PRSV), since, in the case of these and similar promoters, transcription is performed by the DNA-dependent RNA polymerase of the host cell. Also the SP6, T3 or T7 promoters can be used provided that the cell has beforehand been transformed with genes encoding SP6, T3 or T7 RNA polymerase molecules which are either inserted into the chromosome or remain episomal. Expression of these (SP6, T3, T7) RNA polymerase-encoding genes is dependent on the host cell DNA-dependent RNA polymerase.
According to the present invention, the exogenous cDNA insert comprises the coding sequence for a desired product, suitable a biologically active protein or polypeptide, e.g. an immunogenic or antigenic protein or polypeptide, or a therapeutically active protein or polypeptide.
In accordance with another aspect of the invention, the exogenous cDNA insert comprises a sequence complementary to an RNA sequence, such as an anti-sense RNA sequence, which antisense sequence can be administered to an individual to inhibit translation of a complementary polynucleotide in cells of the said individual. The exogenous cDNA may also comprise additional sequences, such as a sequence complementary to an RNA sequence which is a self-cleaving ribozyme sequence. Suitably, the cDNA insert of the present invention is comprised of an integral sequence but the occurrence of interrupting viral sequence(s) is not precluded.
As per definition, in vitro means a process performed outside a living organism as opposed to in vivo which means that a process is performed inside a living organism. According to the present invention, a living organism is intended also to include living cells, such as cultured eukaryotic or prokaryotic cells.
In accordance with the present invention xe2x80x9ctransformationxe2x80x9d is intended to mean introduction in general of exogenous polynucleotides sequences into the interior of a cell, eukaryotic or prokaryotic, and the exogenous polynucleotide sequence may remain extrachromosomal (episomal) or may be stably integrated into the cell genome. The mode of transformation is not crucial, but any means, known at present or that may be developed in the future, can be used according to the invention.
The present alphavirus cDNA vector is based on cDNA, which is complementary to an alphavirus RNA sequence. Once transcribed from the cDNA under transcriptional control of the heterologous promoter, the alphavirus RNA will be able to self-replicate by means of its own replicase and thereby amplifying the copy number of the transcribed recombinant RNA molecules. The replicase will also cap the 5xe2x80x2 ends of each of these molecules. As a result of these events, high levels of expression of the heterologous insert cDNA sequences can be obtained in vivo in the animal or human individual
Contrary to WO 92/10578, the present invention is directed to a cDNA construct, which can be introduced and transcribed per se in animal or human cells, rather than to RNA constructs. However, once the cDNA has been transcribed into RNA subsequent replicative steps and gene expression are in principle the same as described for vectors in WO 92/10578. The disclosure of WO 92/10578 is included in this application by reference thereto.
In the following, a suitable embodiment of the present invention is disclosed to illustrate the present invention without restriction thereof. According to this embodiment, a cDNA molecule comprising nucleotides 1 through 8265 of pSFV1 (WO 92/10578 and Bio/Technology, Volume 9, pages 1356-1361, 1991), containing a heterologous insert e.g. encoding a viral antigen (such as the influenza hemagglutinin protein, influenza nucleoprotein, HIV-1 envelope protein, HIV-1 gag-pol, HIV-1 nef), or a therapeutic protein (such as human growth hormone, interleukin-2, erythropoietin, or factor VIII), or a ribozyme or anti-sense RNA, functionally inserted downstream of the alphavirus subgenomic promoter, is cloned under the CMV promoter in a plasmid in such a way, that the expression of the cDNA insert is driven by the CMV promoter. Downstream from the 3xe2x80x2 end of the cDNA insert, a transcription termination signal (e.g. derived from SV40) is positioned to stop transcription. When this plasmid is transferred into an animal cell, the CMV promoter will guide the transcription of the cDNA insert with its heterologous insert to form one long RNA molecule. This will be transported to the cytoplasm where it is used as mRNA for translation of the nonstructural replicase proteins of the SFV vector. Since the initially transcribed RNA molecule carries sequences required for its replication, the replicase proteins will subsequently initiate replication of the RNA molecule to minus strand intermediates. Subsequently, the SFV replicase will use the internal 26S promoter on the minus strand of the recombinant RNA molecule to produce the messenger RNA molecule encoding the heterologous protein or giving rise to RNA. The RNA replicative events have been described in more detail in WO 92/10578.
Efficient replication of the alphavirus genome is known to require proper 5xe2x80x2 and 3xe2x80x2 ends.
Thus, according to a suitable embodiment of the invention a self-cleaving ribozyme sequence is inserted within the end region of the cDNA insert. This ribozyme molecule is positioned at the 3xe2x80x2 end of the alphavirus genomic sequence in such a way that it, when cleaved, will generate the proper alphavirus 3xe2x80x2 end. Accordingly, when the primary transcript has been made and elongation terminated at the transciption stop signal (e.g. SV40), the ribozyme sequence, carried within the 3xe2x80x2 end domain of the transcipt will self-cleave to generate the proper 3xe2x80x2 end.
In accordance with another embodiment of the present invention an exact 3xe2x80x2 end of the alphavirus RNA molecule is achieved with use of linearized cDNA molecules for the initial transfection. Thus, synthesis from the promoter will result in run-off transcription giving molecules with proper 3xe2x80x2 ends.
In accordance with a suitable embodiment of the invention, in the recombinant cDNA the alphavirus derived cDNA molecule regions comprise sequences complementary to a 5xe2x80x2 terminal portion, the coding region(s) for non-structural proteins required for RNA replication, the subgenome promoter region and a 3xe2x80x2 terminal portion of said viral RNA.
Another embodiment of the invention is concerned with a recombinant cDNA, wherein the exogenous cDNA sequence encodes a foreign polypeptide or gives rise to RNA, said sequence being integrated into the cDNA complementary to the alphavirus subgenomic RNA substituting one or more nucleotides thereof.
A further embodiment of the invention is related to a recombinant cDNA, wherein the exogenous cDNA sequence encodes a foreign polypeptide or gives rise to RNA, said sequence being integrated into the alphavirus subgenomic RNA without substituting any nucleotides thereof.
A broad range of host cells of animal (including human) origin can be used according to the present invention. Such host cells can be selected from avian, mammalian, amphibian, insect and fish cells. Illustrative of mammalian cells are human, monkey, hamster, mouse and porcine cells. Suitable avian cells are chicken cells.
The present cDNA molecules can be used for the treatment of infectious disease, cancer or metabolic disorder or other types of deficiencies in animals and humans. They can also be used for prophylactic treatment or vaccination of animals or humans to prevent infectious disease or cancer. The molecules constitute themselves pharmaceuticals, which operatively code for a biologically active polypeptide or give rise to biologically active polynucleotides, such as antisense RNA, and can be administered directly as such or in combination with other compounds into the animal or human for the expression of the desired sequences.
According to one aspect of the invention, said molecules are used as naked plasmid cDNA molecules and can be administered by intramuscular, intradermal, intranasal, epidermal, mucosal, intravenous route or any other route.
In accordance with another aspect of the invention, the present molecules are mixed with lipids or other compounds to enhance delivery (Trends in Biotechnology,
Volume 11, pages 211-215, 1993). The cDNA can also be linked to other carrier molecules which bind to cellular receptors for uptake into cells (Trends in Biotechnology, Volume 11, pages 202-205, 1993). Such combined strategies can be used by any route of administration. The naked cDNA can also be administered by the means of particle bombardment (Current Opinion in Biotechnology, Volume 4, pages 583-590, 1993).
Moreover, the present cDNA polynucleotide can be administered as part of the genome of another virus, such as a retrovirus. It has been shown earlier that the alphavirus replicase, when produced from a heterologous viral promoter can efficiently perform replication of alphavirus RNA molecules (Journal of Virology, Volume 65, pages 6714-6723, 1991; Virus Research, Volume 23, pages 209-222, 1992).
The present invention is also related to a method, wherein the present cDNA, or the cultured cells comprising this cDNA, is (are) introduced into an animal to produce a product by expression of said cDNA, which product can be recovered from the animal and which product has no effect, which is beneficial to the individual animal, wherein it is produced. Suitably, the expression product is secreted into a body fluid, such as blood, milk or ascites, and is recovered by collection of said fluid. In accordance with one embodiment of this method, the expression product has therapeutic or prophylactic activity and is recovered in a body fluid, such as milk.
In another embodiment of this method, expression of a cDNA, comprising exogenous cDNA coding for an immunogenic or antigenic protein or polypeptide, is achieved and elicits an antibody response, antibodies being collected from the animal in a body fluid, such as whole blood, serum or ascites.
According to a further embodiment of this method, the cDNA comprises exogenous cDNA coding for an antigenic determinant, antigens or immunogens being produced by expression of the cDNA and recovered from the animal in a body fluid, such as whole blood or serum.